Project Summary/Abstract: Craniofacial bone grafts are used to treat over 200,000 patients in the United States annually. Autograft, the current standard of treatment, has multiple drawbacks including donor-site morbidity and lack of available tissue. A promising alternative is the combination adipose-derived stem/stromal cells (ASCs) with osteoinductive biomaterial scaffolds that can be 3D-printed to mimic the native geometry of the defect. ASCs can be readily obtained in high yields from non-invasive procedures and have been shown to mineralize robustly in vitro, leading to their utility as a stem cell source. Despite these promising characteristics, ASCs have demonstrated limited ability to regenerate bone in vivo. A predominant hypothesis is that the hypoxic environment following implantation may lead to massive cell death; however, in preliminary in vitro experiments, I have observed excellent (>70%) ASC survival in severely hypoxic culture, but during in vitro osteogenic differentiation, Runx2 expression and alkaline phosphatase (ALP) activity, two common markers for osteogenesis, are inhibited by hypoxia. Additionally, through the use of a novel strategy for delivering oxygen to cells seeded in 3D scaffolds, I have demonstrated that providing oxygen in situ to transplanted ASCs doubled the amount of bone formed in vivo in a murine ectopic bone formation model. Thus, the major premise of this proposal is that the reduced in vivo bone formation by ASCs is due to the direct inhibition of ASC osteogenesis by hypoxia. The objective of the proposed study is to investigate the interplay between hypoxia and ASC osteogenesis, overcoming several major limitations in the field. First, ASC osteogenesis in hypoxia will be studied quantitatively using a 3D in vitro model of bone formation Previous literature examining the impact of oxygen on ASC differentiation produced conflicting results, because they relied on qualitative metrics of osteogenesis. In Specific Aim 1, I will use a 3D in vitro model of bone formation to quantify changes in mineralization, tissue microarchitecture, metabolism, and gene expression due to hypoxia. Extensive gene expression data may allow us to identify novel mediators of oxygen-dependent ASC osteogenesis. Next, in Specific Aim 2, I will investigate the interplay between hypoxic signaling and ASC osteogenesis using gain-of- function and loss-of-function studies using novel non-viral polymeric nanoparticles and oxygen-releasing scaffolds. First, I will determine whether the effect of hypoxia on ASC osteogenesis can be simulated by upregulating hypoxia-inducible factor-1? (HIF-1?) downregulating HIF-1?. Third, I will determine the effect of oxygen release on ASC osteogenesis. Finally, in Specific Aim 3, I will study the effects of HIF-1? gain-of- function and loss-of-function and oxygen delivery on ASC osteogenesis in a calvarial defect model. The results of these studies will deepen the understanding between hypoxia and osteogenesis and inform efforts to improve ASC osteogenesis in vivo.